Method for the biochemical isolation of l-menthol

ABSTRACT

Method for the biochemical isolation of l-menthol. Specifically, an improved method for the optical resolution of racemic menthol utilizing an enzyme, carboxylic ester hydrolase, produced by the action of a micro-organism belonging to the class of Penicillium, Gliocladium, Trichoderma, Geotrichum, Aspergillus, Pullaria, Fusarium, Absidia, Cunninghamella, Rhizopus, Actinomucor, Chlamyclomucor, Mucor, Gibberella, Streptomyces or Bacillus. The l-menthol may be utilized in perfumes, medicines, etc.

United States Patent Inventors Tatsuo Moroe Tokyo; Satohlka Hattori,Yokohama-shl; Aklra Kolnatsu, Tokyo; Yum Yamaguchl, Yokohama-sh], all 0!Japan Appl. No. 783,126

Filed Dec. 11, 1968 Patented Sept. 21, 1971 Assignee Takasago PerfumeryCo., Ltd

Tokyo, Japan METHOD FOR THE BIOCHEMICAL ISOLATION OF I -MENTHOL Field 0!Search 195/2, 30, 3

Assistant Examiner-Max D. Hensley Attorney-Sughrue, Rothwell. Mion, Zinn& Macpeak ABSTRACT: Method for the biochemical isolation of I- menthol.Specifically, an improved method for the optical resolution of racemicmenthol utilizing an enzyme, carboxylic ester hydrolase, produced by theaction of a micro-organism belonging to the class of Penicillium.Gliocladium, Trichoderma, Geotrichum, Aspergillus. Pullaria, FusariumAbsidia, Cunninghamella, Rhizopus. Actinomucor. Chlamyclomucor, Mucor,Gibberella. Streptomyces or Bacillus The I-menthol may be utilized inperfumes, medicines, etc.

METHOD FOR THE BIOCHEMICAL ISOLATION OF ETL QI:

BACKGROUND OF THE INVENTION 1. Field of the Invention The presentinvention relates to a method for the biochemical isolation ofl-menthol.

2. Description of the Prior Art From the stereochemical configuration ofmenthol, four stereo isomers can exist, that is, menthol, isomenthol,neomenthol and neoisomenthol. Each of these stereo isomers has opticalisomers (dextrorotatory d and levorotatory 1 forms) so there are eightoptical isomers in all. Among these isomers l-menthol is one of the maincomponents of natural mint oil, and illustrates the strongest refreshingactivity so that it is widely used in perfumes and in medicines.

The l-menthol may be produced by a number of methods. For example, thecrystal of l-rnenthol cam be isolated from natural mint oil by cooling.Synthetically, I-menthol can be obtained from l-menthone which iscontained in natural mint oil by reduction with metallic sodium. On anindustrial scale, menthol is produced from d-citronellal, which is acompound of citronella oil, by cyclization and hydrogenation. But asthese natural raw materials (l-menthone and d-citronellal which haveoptical activities are available in a very limited supply, it isdesirable to synthesize l-Inenthol from low priced industrial chemicals.

When l-menthol is synthesized from optically inactive raw materials, oneimportant problem is to separate only optically active l-menthol from amixture of d l-menthol isomers, because the simultaneous formation ofall of the d l-isomers (d l-menthol, d l-isomenthol, d l-neomenthol andd neoisomenthol) is unavoidable. Many attempts to separate menthol havebeen made in the past, but a successful industrial process has not yetbeen realized. For this reason, d lmenthol is actually utilized in manyinstances without complete product purity.

The main industrial process for d l-menthol synthesis consists of thehydrogenation of thymol and the heat equilibration of the resultantmixture of d l-menthol isomers to the following composition: dImentholabout 70 percent; d lisomenthol-about percent; :1l-neomenthol-about it) percent; and d l-neoisomenthol-trace. Further,the ratio of these isomers would vary with reaction conditions makingthe product composition extremely variable.

Another method of d l-menthol synthesis consists of making a mixture ofd l-menthol isomers. the components of which are primarily d l-menthol(about 70 percent), :1 l-isomenthol and d l-neomenthol, by cyclizationand hydrogenation of d citronellal, with d I-menthol being separatedtherefrom.

The hitherto known methods for the optical resolution of the thusobtained d l-menthol involve the optical isolation of this compound fromits phthalic acid semiesters, I-menthoxy acetic acid ester and dl-camphoric acid ester. These methods are too expensive and involve aprocedure which is too complicated to be used on an industrial scale,and are therefore only utilized as laboratory processes.

SUMMARY OF THE INVENTION It has been discovered that an optically activel-menthol may be biochemically isolated by a process which comprises theoptical resolution of an organic carboxylic acid ester of d lrnenthol,or by the optical resolution of the organic carboxylic acid esters of amixture of d l-menthol isomers (d l-menthol, d l-isomenthol, dl-neomenthol and d l-neoisornenthol). An enzyme, or number of enzymes,which comprise carboxylic ester hydrolases which have been produced bythe action of microorganisms from the class consisting of Penicillum,Gliocladium, Trichoderma, Geotrichum, Aspergillus, Pullularia, Fusarium,Absidia, Cunninghamella, Rhizopus, Actinomucor, Chlamydomucor, Mucor,Gibberella, Streptomyces and Bacillus, may be utilized.

It is an object of the present invention to provide a method for thebiochemical isolation of l-menthol.

It is another object of the present invention to provide a novel andindustrializable method for the optical resolution of d l-menthol or amixture of d I-menthol isomers containing d menthol.

It is still another object of the present invention to provide thepractical and useful method for the production of I- menthol.

It is the other object of the present invention to provide industriallyoptical active l-menthol which is useful for the production of perfumesand medicines from thymol.

DESCRIPTION OF THE PREFERRED EMBODIMENTS As a result of manyinvestigations into the asymmetric hydrolysis of esters of d l-mentholisomers with organic carboxylic acids, such as d l-menthyl ester, dl-neoisomenthyl ester and d l-isomenthyl ester, by means of carboxylicester hydrolase, which is obtained from some types of micro-organisms,as bacteria and mold, the present inventors have found that d l-menthylester and d l-isomenthyl ester were hydrolyzed, respectively, intol-menthol, l-isomenthol; that the d-form was never decomposed; that thed l-neomenthyl ester and the d l-neoisomenthyl ester were not attackedby carboxylic ester hydrolase; and finally that this fact offers asolution to the problem of obtaining I-menthol from the organic car'boxylic acid esters of d l-menthol and organic carboxylic acid, ormixtures of esters of the d I-menthol isomers which contain d l-mentholtherein. Further, the present method is applicable on an industrialscale to a much greater extent that former methods of optical resolutionwhich can be used only in the laboratory.

The present invention is concerned with a method for the biochemicalisolation of l-menthol which comprises treating an organic carboxylicacid ester of d l-mcnthol or mixture of this ester with organiccarboxylic acid esters of d l-menthol isomers containing d l-mentholtherein (wherein the organic carboxylic acid is formic acid or a fattyacid of the general formula: RCOOH, and R is an alkyl group or analkenyl group of from I to 21 carbon atoms) with an enzyme, carboxylicester hydrolase, which may be produced by the action of micro-organismsbelonging to the group penicillium, Ciliocladium, Trichoderma,Geotricum, Aspergillus, Pullularia, Fusarium, Absida, Cunninghamella,Rhizopus, Actinomucor, Chlamydomucor, Mucor, Gibberella, Streptomycesand Bacillus which have been separated from their cell bodies and theirculture medium or which may alternatively be directly used in the formof their cell bodies or culture mediums containing the said enzyme;carboxylic ester hydrolase. The isomer-enzyme composite is subjected toasymmetric hydrolysis, and then an optically active l-menthol isseparated.

The screening and selection of the micro-organisms (bacteria and mold)useful in producing carboxylic ester hydrolase has been practiced bydetermining the asymmetric hydrolysis activity of d l-menthyl ester(hydrolysis ration and angle of rotation). Finally micro-organismsproducing the required enzyme, possessing the activity of carboxylicester hydrolase, have been selected from the group consisting ofPenicillium, Gliocladium, Trichoderma, Geotrichum, Aspergillus,Pullularia, Fusarium, Absidia, Cunninghamella, Rhizopus, Actinomucor,Chlamydomucor, Mucor, Gibberella, Streptomyces and Bacillus. The resultsof a determination of the asymmetric hydrolysis ratio according to thisenzyme, carboxylic ester hydrolase, are set forth in Table l. The testfor the asymmetric hydrolysis of d l-menthyl acetate was performed asfollows: microorganisms fested were cultured under agitation for 2 daysat a temperature of 27 C. in g. of a media comprising glucose, phosphateand a peptone medium [7 g. of glucose, phosphate, peptone, 5 g. of yeastextract, 15 g. of glucose, 2 g. of agar and I000 g. of water) for molds,and a standard medium l0 g. of peptone, 4 g. of meat extract, l g. ofglucose, and 2 g. of agar) for bacteria. Then, d l-menthyl acetate wasadded to the culture medium at a concentration as shown in table 1 withagitation (shaking) being continued at a temperature of 27 C. for 2days. The medium was subjected to steam distillation, the distillatebeing analyzed by gas chromatography means and the hydrolysis ratio(l-menthol XlOO/d l-menthyl acetate +l-menthol) was calculated from thepeak area of the gas chromatograms.

TABLE I Organism Concentration of Hydrolysis ratio dbmenthyl acetate(weight percent) Penicillum frequentans 2 27.4 PentcillumspinuIo-ramigenum l 14.8 Gliocladium roseum I I2]! Gliocladium SP 20.4Tnchodermn viride 2 60.2 Trichodermn komngi l $7.2 Trlchuderma SP 9484 280.6 Ueotrichum Candidum I 68.3 Ueotrichum SP 1 2 L6 Aspergillus flavusvar aspcr I 55.2 Aspergillui aponicus l 24.0 Pullularia pullulans 2 46.2Pullularla SP 2 33.6 Fusarium roseum 2 I91] l-usariurn graminearum 118.8 Absidla glauca var paradoxa 2 98.4 Absidia hyalospora 2 98.6(unninghamella ellegans 2 76.4 Cunninghamella SP I 20.0 Rhlzopus peka 169.1 nigticans I 40.0 Actinomucor repens 2 26.8 Actinomucor SP I [2.3Chlamydomucor javantcus t 10.4 Chlamytlornucor SP 1 I56 Mucur hiemalis I25.8 Mucor gnseocyanus I 15.5 Urbberella l'ujilruroi I 42.5 (iihberellasanhiinelii l l5.5 itrcptomyces griseus l llHl Streptomyces SP 1 l5.(]Bacillus sublilis var niger 5 97.5 HitCIlllN mesentericus I 20.4Hucillus pumilus Z 3 l .3

The classification of the fungi was based on Ainsworth 8.. Bisby'sDictionary of the Fungi," and that of bacteria was based on Bergey'sManual of Determinative Bacteriology, 7th edition."

As the organic carboxylic acid used to produce the organic carboxylicacid ester of d I-menthol, formic acid and organic carboxylic acidsofthe general formula RCOOH were used (wherein R is an alkyl or analkenyl group having from I to 2l carbon atoms). Examples of the acidare, for example, acetic acid, propionic acid, butyric acid, caproicacid, caprylic acid, capric acid, lauric acid, myristic acid, palmiticacid, stearic acid, acrylic acid, oleic acid, and erucic acid. Formicacid, acetic acid, propionic acid and myristic acid are best suited foruse in the present invention of these carboxylic acids, especiallyacetic acid since it is lowest in cost and gives a high asymmetrichydrolysis ratio.

The most important, and characteristic, aspect of the present inventionis the enzymatic treatment of an organic carboxylic acid ester of dI-menthol, or a mixture of the organic carboxylic acid esters ofdl-menthol isomers containing the organic carboxylic acid ester ofdl-menthol with carboxylic ester hydrolase produced by the action ofmicro-organisms as described above and utilizing the hydrolase toselectively hydrolyze the esters of l-menthol and I-isomenthol.

In the present invention, hydrolysis of the esters of I- mcnthol andl-isomenthol was carried out not only by shaking in contact with thegrowing micro-organisms or the intact cells thereof, but also by mixingwith the culture medium free from living cells and with the cell-freeextracts of the above described micro-organisms.

ln carrying out this process, the preferred reaction temperature is20-45 C., most preferably 25-30 C., in order to minimize inactivation ofthe enzymes by heat denaturation. The reaction time is about 24-48 hoursat 25-30 C., but will be shorter at higher temperatures. The amount ofenzyme utilized in crude form (for example, ex. 4), is about 0.1-0.5percent based on the reaction mixture.

The amount of the organic carboxyclic esters of the mixture of dl-menthol isomers, is preferably in the range of about 1-10 percent ofthe reaction mixture, At higher concentrations, the rate of hydrolysismay be decreased. However, even at higher concentrations, the reactionrate may be increased by using a larger amount of the enzymes.

In general, as an economical amount of esters, there may be used from lto l0 percent of the esters per 0.1-0.5 percent of the crude enzymesolution or the corresponding culture fluid.

Among the foregoing d l-menthol isomers, d neoisomenthol can beconverted into other menthol isomers by heat isomerization with ahydrogenating catalyst, thus a d I-neoisomenthol-free mixture can easilybe obtained.

Upon performing the carboxylic ester hydrolasc described on the mixtureof organic carboxylic acid esters of these three d I-menthol isomers,l-menthol and Lisomenthol are released according to the asymmetrichydrolysis, and the other esters remain.

For example, in the case of the organic carboxylic acid esters of dl-menthol, only the I-form is hydrolyzed. The dform is not affected andremains unchanged.

Separation of said Lmenthol from the reaction mixture can easily beperformed by a fractional distillation.

Further, l-menthol (m.p. 42-43 C., b.p. 2l6.5 C.) and I- isomenthol(m.p. 82.5 C., b.p. 2l8.6 C.) may simply be separated by rectification,a derivative formation (cyanoacetic acid ester, monochloroacetic acidester, monophthalic acid ester, etc.), recrystallization,chromatography, etc. The following examples will serve to set forth thepreferred embodiment of the present invention.

EXAMPLE I Asymmetric hydrolysis of d I-menthyl acetate by Absidiahyalospora was carried out as follows:

Twenty Kg. of the heretofore described glucose, phosphate, peptonemedium was poured into a 30 ljar-fermentor, after heat sterilization(l20 C., l5 min.) 500 g. of an Absidia hyalospora starter (cultured inthe same medium at 27 C., for 2 days while being shaken into it) wasinoculated into the fermentor, and the mixture was cultured for 24 hoursat 27 C., an airflow of 5 l/min., and at a stirring rate of 200 rpm. Twohundred g. of d I-menthyl acetate were added to the culture fluid, andthis mixture was kept at 27 C., with stirring at 300 r.p.m. for 24hours. The fermented solution was steam distilled and the distillate wasextracted with toluene and finally concentrated to give I g. ofaconcentrated oil.

From the results of a gas chromatographic analysis (inert solid support:celite, stationary liquid: silicon 20 percent, column dimensions: 2 m.0.3 cm., temperature l55 C., carrier gas: helium, pressure at entrance:1 kgjcm the content of I-menthol was determined to be 49.5 percent.

By fractional distillation of the oil, l-menthol, b.p. 98 C./l0 mm, Hg:[a]"=50 C. (C=3: in ethanol) 75 g. and d-menthyl acetate, b.p. 109 C./l0mm. Hg, :[a]=+80 C. (C=4: in ethanol) g, were determined.

EXAMPLE2 Asymmetric hydrolysis of d l-menthol isomers by Trichodermaviride was carried out as follows.

Twenty Kg. of the heretofore-described glucose.phosphate.peptane mediumwas poured into a 30ljar-fermenter. After heat sterilization, there wasinjected thereinto 500 g. of a Trichoderma viride starter at 28 C. Thestarter was cultured for 24 hours, at 27 C. under an airflow of 5I/min., and d stirring at 300 rpm, 200 g. of the acetates of d mentholisomers containing dl-menthol (d l-neornenthol 5.8 percent, d l-menthol63,8 percent, d l-isomenthol 30.4 percent) were added, and the solutionwas held at 28 C., stirring it at 300 r.p.m., for 24 hours. After asteam distillation of the fermented solution, the distillation oil wasextracted with toluene to yield 173 g. of the concentrated oil. From theresults of a gas chromatographic analysis (same conditions as in exampleI the d l-neomenthol acetate was determined to be 5 percent, thed-menthyl acetate d-isomenthyl acetate 48.5 percent, the I-menthol 28.8percent, and the l-isomenthol l7.4 percent.

One hundred g. of the obtained oil was subject to a chromatographicanalysis by utilizing alumina (lkg.) After developing with n-hexane, anelution with N-hexane (2!) was performed to give 50 g. of a mixture of dI-neomenthol acetate, d-menthyl acetate and d-isomenthyl acetate. Afurther elution with 2! of ethyl acetate was performed to give 45 g. ofa mixture of l-menthol (62.3 percent) +l-isomenthol (37.7 percent).

This final mixture was esterified with monochloroacetic acid andrecrystallized from methanol to separate a pure menthyl monochloroaceticacid ester [a]"=-77.5 C. (in ethanol), m.p. 38C.

By hydrolizing this ester with an aqueous solution of caustic soda,about 23 g. of I-menthol [a]'"=50 C. (in ethanol) was obtained.

EXAMPLE 3 Separation of carboxylic ester hydrolase from the parentmicro-organisms and the asymmetric hydrolysis of d l-menthyl acetateusing the carboxylic ester hydrolase was carried out as follows.

Trichoderma SP 9482 was cultured in lkg. of the heretofore-describedglucose.phosphate.peptone medium, at 27 C., for 3 days with shaking.Ammonium sulfate was added to 900 g. of a filtrate of the culture medium(at a low temperature) at a 70 percent saturation degree. After allowingthe solution to stand for 12 hours, crude enzymes were separated by ahighspeed centrifugal separator from the solution and freeze dried toyield 3 g. of the crude enzyme.

Three g. of the crude enzyme were dissolved in 800 g. of water and 200g. of a 2 percent polyvinyl alcohol emulsion. Twenty g. ofd l-menthylacetate were added, and the solution shaken; for 2 days at 27 C. After asteam distillation of the reaction mixture, the distillate was extractedwith ether, dried, and concentrated to obtain l9 g. of oil. From theresults of a gas chromatographic analysis (inert solid support: celite,sta tionary liquid: silicon percent, column dimensions: 3m. X0.3cm.,temperature: 180 C., carrier gas: helium, pressure at entrance: IkgJcm?) the content of I-menthol was determined to be 43.3 percent.

By fractional distillation of the oil, l-menthol [(a)=50 (C.=4, inethanol), b.p. 98 C./l0 mm. Hg. 1, was obtained in a yield of 7.3 g.

EXAMPLE 4 Asymmetric hydrolysis of d l-menthyl acetate by Bacillussubtillus var Niger was carried out as follows.

Bacillus subtillus var Niger was cultured in lkg. ofa general standardmedium at 27 C. for 2 days, with shaking. Thirty g. of d I-menthylacetate was added, and the solution was shaken at 27 C. for 2 days.After a steam distillation of the fermented solution, the distillate wasextracted with ether and concentrated. The yield was 27 g. From theresults ofa gas chromotographic analysis, the content of l-menthol wasdetermined to be 50.3 percent.

The concentrated oil was applied to 350 g. of chromatographic alumina toobtain l4 g. of d-menthyl acetate [(01)' ='82 (C.=3, in ethanol) byelution with n-hexane. Twelve g. of I-menthol (a)"=49 (C.=5, in ethanol]were obtained by further elution with benzene.

EXAMPLE 5 Asymmetric hydrolysis of d l-menthyl acetate by the liquidculture medium of Geotrichum candidium was carried out as follows.

Geotrichum candidium was cultured in a wheat bran medium 10 g. of wheatbran g. of water) at 27 C. entrance: 3 days, with shaking. 2 g. of dI-menthyl acetate was added, and the solution shaken at 27 C. for 2days. After a steam distillation of the fermented solution, thedistillate was extracted with either and dried with magnesium sulfate toobtain [.8 g. of oil, by concentration. From the results of a gaschromatographic analysis (inert solid support: celite, stationaryliquid: silicon 20 percent, column dimension: 3m.=0.3cm., temperature:180 C., carrier gas: helium, pressure at entrance: 1 kg./cm), thecontent of I-menthol was determined to be 40.2 percent.

By applying the above to 30 g. of alumina utilized in chromatography,there was obtained 1 g. of d-menthyl acetate by elution with n-hexane,and 0.7 g. of l-menthyl la]=50 (in ethanol) by further elution withbenzene.

EXAMPLE 6 Asymmetric hydrolysis of d Imenthyl formate was carried out asfollows.

Two g. of dl-menthyl formate was phosphate. to I00 g. of the liquidculture medium of Trichoderma SP 9484 (example 3) which was prepared byshaking at 27 C. for 2 days in the heretofore described glucose.phosphate. peptone medium. The composite was maintained at 27 C. for 2days under agitation shaking. After a steam distillation of thefermented solution, the distillate was extracted with ether to obtain aconcentrated oil (yield: 1.0 g.). g.) From the results of a gaschromagraphy analysis of the concentrated oil, the content of l-mentholwas l7.45 percent. Further treatment by alumina chromatography as inexample 5 yielded 300 mg. of I-menthol [a]""=-45C.

EXAMPLE 7 This example follows the basic procedure of example 6 with theexception that 2 g. of dl-menthyl propionate was employed instead of 2g. of the dl-menthyl formate of example 6. The yield of the concentratedoil from the dI-menthyl propionate was 1.8 g. and the l-menthol contentthereof was 49.98 percent. Chromotographic separation of the dlmenthylpropionate give 800 mg. of l-menthyl, [a] =49 C.

EXAMPLE 8 Asymmetric hydrolysis of dl-menthyl Trichoderma viride wascarried out as follows.

Trichoderma viride was cultured in 1 kg. of the heretofore describedglucose.phosephate.peptone medium at 27 for 2 days with shaking. Twentyg. of dI-menthyl mystrate were added and the solution further shaken at27 C. for three days. After a steam distillation of the fermentedsolution, the distilled I-menthol was extracted with ether, dried withmagnesium sulfate and concentrated to yield 1.7 g. of l-menthol,[a]*=30" (C.=6, in percent of ethanol).

What is claimed is: l. A method for the biochemical isolation ofl-menthol which comprises:

asymmetrically hydrolyzing a member selected from the group consistingof an organic carboxylic acid ester of d!- menthol and a mixture oforganic carboxylic acid esters of dI-menthol isomers containingdlmenthol, wherein the organic carboxylic acid utilized to form saidester is selected from the group consisting of formic acid and mystrateby fatty acids of the general formula RCOOH, wherein R is a memberselected from the group consisting of an alkyl group and an alkenylgroup having from 1 to 2] carbon atoms, said asymmetric hydrolysis beingperformed by an enzyme, carboxylic ester hydrolase, which has beenproduced by the action of micro-organisms belonging to the classconsisting of Pencillium, Gliocladium, Trichoderma, Geotrichum.Aspergillus, Pullularia, Fusarium. Absidia, Cunninghamella, Rhizopus,Actinomucor, Chlamydomucor, Mucor, Gibberella, Streptomyces andBacillus; and

separating optically active Lmenthol from the enzyme reaction mixture.

2. A process as in claim 1, wherein said carboxylic acid is acetic acid.

3. A process as in claim I, wherein said hydrolyzation is conducted at atemperature of from about 20 to about 45 C.

4. A process as in claim I, wherein the amount of enzyme utilized isfrom about 0.1 to 0.5 percent, by weight. based on the enzyme reactionmixture.

5. A process as in claim 1, wherein the amount of organic carboxylicester is in the range of from about 1 to about [0 percent by weight,based on the reaction mixture.

6. A process as in claim I, wherein said enzyme is utilized in the formof actively growing micro-organism cells from the defined class.

7. A process as in claim 1, wherein said enzyme is utilized in the formof a cell free extract produced from said micro-organisms.

2. A process as in claim 1, wherein said carboxylic acid is acetic acid.3. A process as in claim 1, wherein said hydrolyzation is conducted at atemperature of from about 20 to about 45* C.
 4. A process as in claim 1,wherein the amount of enzyme utilized is from about 0.1 to 0.5 percent,by weight, based on the enzyme reaction mixture.
 5. A process as inclaim 1, wherein the amount of organic carboxylic ester is in the rangeof from about 1 to about 10 percent by weight, based on the reactionmixture.
 6. A process as in claim 1, wherein said enzyme is utilized inthe form of actively growing micro-organism cells from the definedclass.
 7. A process as in claim 1, wherein said enzyme is utilized inthe form of a cell free extract produced from said micro-organisms.